39
J. Anat. (1991), 178, pp. 39-43 With 6 figures Printed in Great Britain
Relationships between lymphoid nodules and lymph sinuses in lymph nodes: a study in horses TREVOR J. HEATH* AND STEPHEN A. NIKLES
Department of Anatomy, University of Queensland, Queensland 4072, Australia
(Accepted 10 April 1991) INTRODUCTION
Secondary lymph nodules traditionally appear in the outer cortex, separated from the subcapsular sinus by a variable amount of cortical tissue (Banks, 1986; Raviola, 1986; Brown, Dellmann & Nicander, 1987). Studies in small laboratory animals have revealed that antigenic materials in the lymph entering the subcapsular sinus may rapidly penetrate this cortical tissue and reach the nodule (Nossal & Ada, 1971; Szakal, Holmes & Tew, 1983; Tew, Mandel, Phipps & Szakal, 1983). However, there has been no suggestion from any animals that other sinuses within the node are involved in transport to or from the nodules. Recent studies have shown that the sinus network in horse lymph nodes is more extensive than in other species (Heath & Perkins, 1989; Nikles & Heath, 1991 b). The aim of this study was to define the morphological relationships between these sinuses and the nodules. The study was undertaken using Microfil and methacrylate casts, and by light and electron microscopy.
MATERIALS AND METHODS
Eleven female and 9 male horses, 6-48 months old, were used. All were killed either with an overdose of pentobarbitone, or by humane shooting at a local knackery. These horses were also used either by Heath & Perkins (1989) or by Nikles & Heath (1991 a, b). Up to 10 nodes were taken from each horse, and these were mainly from the following lymph centres: cranial mesenteric, caudal mesenteric, and superficial
inguinal. Methods described by Spalding & Heath (1987) and Nikles & Heath (1991 a) were used to cannulate afferent lymphatic vessels, and to inject either Microfil Rubber Injection Compound (Canton Biomedical Products, Boulder, Colorado), or Batsons 17 Anatomical Corrosion Compound (Paul Valley Industrial Park, Warrington, Pennsylvania) into the lymph sinuses of the node. Methods described by Heath & Perkins (1989) and Nikles & Heath (1991 b) were used to prepare tissues for light microscopy and for scanning electron microscopy. A Philips 505 scanning electron microscope was used. *
Correspondence to Professor T. J. Heath.
40
T. J. HEATH AND S. A. NIKLES RESULTS
The number of secondary nodules within each node varied widely; in some nodes they were sparse, whereas in others nodules were distributed throughout the cortex (Figs 1, 2). There did not appear to be any constant relationship between the number of nodules and the location of the node. The nodules appeared as clear rounded spaces in nodes which had been filled with Microfil and cleared in methyl salicylate (Fig. 3). Their presence was confirmed by cutting the nodes into slices and examining these under a stereomicroscope, and then by studying histological sections prepared from the surface of the slices. The nodules were examined further using a methacrylate-based cast of the lymph sinuses. When the tissue had been digested and the cast broken open to reveal sinuses within the node, scanning electron microscopy revealed rounded spaces similar to those seen in Microfil casts (Figs 3, 4). Examination of the inner surface of these holes revealed a smooth, curving network of filled sinuses, with variable spaces between them (Figs 4, 5). Other nodes were fixed by perfusion of fixative through an afferent lymphatic, and the structure of this network examined in histological sections and by scanning electron microscopy. Virtually all sections through nodules revealed segments of sinuses in the immediate vicinity (Figs 1, 2, 6). In some cases, the sinus-lining cells abutted the capsule of the nodule, but more commonly they were separated by several layers of cells. The nodules at the periphery were adjacent to the subcapsular sinus (Fig. 2), which covered all the cortex. The other types of sinus which were associated with the nodules included those surrounding trabecule, both superficially and deep within the cortex; sinuses between cortical cords of lymphoid parenchyma and the tubule-like extensions of these sinuses into the cortical parenchyma (Figs 1, 2, 6). Some cortical tubular sinuses, which unlike other sinuses had few luminar processes, were also seen in the vicinity of the nodules (Figs 1, 2). DISCUSSION
Secondary lymphoid nodules have been variously referred to as 'follicles' (Tew et al. 1984; van Rooijen, 1987), 'folliculo-nodules' (Sainte-Marie, Peng & Belisle, 1982; Sainte-Marie & Peng, 1986) and 'germinal centres' (Raviola, 1986). In the horse they are surrounded by a network of lymph sinuses. These sinuses include the subcapsular sinus and its extensions around trabeculae, the tubular and tubule-like sinuses deeper in the cortex, and the sinuses between cord-like projections of cortical tissue (Nikles & Heath, 1991 b). These sinuses become filled when casting material is injected through an afferent lymphatic, indicating that they form an interconnecting network throughout the cortex. This contrasts with the situation in the small laboratory animals which have been used to study the movement of antigenic materials within the lymph node. In these animals, the subcapsular sinus appears to be the only sinus that is regularly found in the immediate vicinity of the nodules. However, Sainte-Marie et al. (1982) illustrated infoldings of this sinus near the nodules as one of the variations that may occur in rat lymph nodes. Where the subcapsular sinus forms a continuous sheet, however, a sinus is adjacent to only a small proportion of the nodule surface (SainteMarie et al. 1982; Sainte-Marie & Peng, 1986; van Rooijen, 1987), and separated from it by a subsinus layer of reticular-type cells (Sainte-Marie & Peng, 1985). It appears
~ .,
Nodules and sinuses in lymph nodes
..40
~ ~ ~
~
~
4
~
~
~
~
..
.
A~~~~A
Figs 1, 2. Photomicrographs of sections, stained with haematoxylin and eosin, of the cortex of the horse lymph node. Secondary nodules are located immediately under the subcapsular sinus, and deeper within the cortex. Sinuses in the vicinity of these nodules include the subcapsular sinus (S), trabecular sinuses (T), sinuses between cortical cords (C), and their tubule-like extensions (asterisks) and tubular sinuses (arrows). Fig. 1 x 90; Fig. 2 x 50.
41
42
T. J. HEATH AND S. A. NIKLES
Fig. 3. Photomicrograph of the cut surface of a lymph node which had been filled with Microfili from lymphatic, showing rounded depressions which correspond to nodules. x 20. 5. Figs 4, Scanning electron micrographs of the broken surface of a cast made by injecting Batsons 17 through an afferent lymphatic, showing a network of sinuses surrounding a nodule. The nodule is centrally placed in Figure 4, and occupies the lower half of Figure 5. Fig. 4 x 100; Fig. 5 x 250. Fig. 6. Scanning electron micrograph of the surface of a nodule (N), and an adjacent sinus, which is associated with a trabecula (T). x 200. an afferent
that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to the region of the nodules (Szakal et al. 1983; Tew et al. 1984). In horses the nodules, some of which are located relatively deep in the node, are closely associated over much of their surface with an interconnecting network of lymph sinuses. Thus antigenic material carried in the lymph from the tissues would be well placed to influence the activity of nodules irrespective of where they occur in the node. The sinuses in the horse could also provide an important pathway for cell migration within the node, and in this way play a similar role to the 'cortical pathways of migration of circulating lymphocytes' along a reticular fibre network within lymph
Nodules and sinuses in lymph nodes
43
nodes in rats, which was described by Sainte-Marie & Peng (1986). However, no studies appear to have been undertaken on the movement of antigenic materials or cells in the lymph nodes of the horse. In view of the differences in node structure between small laboratory animals and the horse (Heath & Perkins, 1989; Perkins & Heath, 1990; Nikles & Heath, 1991 a, b) it would not be surprising if appreciable differences did exist between these animals in the pathways taken by immunologically important elements within the node. SUMMARY
Secondary lymphoid nodules in lymph nodes of the horse are surrounded by a network of lymph sinuses, including the subcapsular sinus and its extensions around tabeculae, tubular and tubule-like sinuses deeper in the cortex, and sinuses between cord-like projections of cortical tissue. The precise role of this close association between sinuses and nodules in the transport of immune complexes, cells and cytokines is not known. We are grateful to Gary Godbold for help with obtaining specimens, the University of Queensland Electron Microscope Centre for help with electron microscopy, and the Queensland Equine Research Foundation for financial support. REFERENCES
BANKS, W. J. (1986). Applied Veterinary Histology, 2nd ed. Baltimore: Williams & Wilkins. BROWN, E. M., DELLMANN, H.-D. & NICANDER, L. (1987). Lymphatic organs. In Textbook of Veterinary Histology, 3rd ed. (ed. H.-D. Dellmann & E. M. Brown), pp. 164-184. Philadelphia: Lea & Febiger. HEATH, T. J. & PERKINS, N. R. (1989). Pathways between lymph vessels and sinuses in lymph nodes: a study in horses. Anatomical Record 223, 420424. NIKLES, S. A. & HEATH, T. J. (1991 a). Pathways of lymph flow from the intestine of the horse. Anatomical Record 229, 521-524. NIKLES, S. A. & HEATH, T. J. (1991 b). Pathways of lymph flow through intestinal lymph nodes in the horse. Anatomical Record, in press. NOSSAL, G. J. V. & ADA, G. L. (1971). Microscopic and electronmicroscopic distribution of antigen in lymphoid organs. In Antigens, Lymphoid Cells and the Immune Response, pp. 107-141. New York: Academic Press. PERKINS, N. R. & HEATH, T. J. (1990). Pathways of lymph flow from the superficial tissues in the legs of horses. Research in Veterinary Science 48, 119-123. RAVIOLA, E. (1986). The immune system. In Bloom and Fawcett, A Textbook of Histology (ed. D. W. Fawcett), pp. 406-435. Philadelphia: Saunders. SAINTE-MAREE, G. & PENG, F.-S. (1985). Evidence for the existence of a subsinus layer of the peripheral cortex in the lymph node of the rat. Cell and Tissue Research 239, 37-42. SAINTE-MARIE, G. & PENG, F.-S. (1986). Diffusion of lymph-carried antigen in the fiber network of the lymph node of the rat. Cell and Tissue Research 245, 481-486. SAINTE-MAREE, G., PENG, F.-S. & BESLISLE, C. (1982). Overall architecture and pattern of lymph flow in the rat lymph node. American Journal of Anatomy 184, 275-309. SPALDING, H. J. & HEATH, T. J. (1987). Pathways of lymph flow through superficial inguinal lymph nodes in the pig. Anatomical Record 217, 188-195. SZAKAL, A. K., HOLMES, K. L. & TEw, J. G. (1983). Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. Journal of Immunology 131, 1714-1727. TEW, J. G., MANDEL, T. E., PHIpps, R. P. & SZAKAL, A. K. (1984). Tissue localization and retention of antigen in relation to the immune response. American Journal of Anatomy 170, 407-420. VAN ROOIJEN, N. (1987). The 'in situ' immune response in lymph nodes: a review. Anatomical Record 218, 359-364.
J. Anat. (1991), 178, pp. 39-43 With 6 figures Printed in Great Britain
Relationships between lymphoid nodules and lymph sinuses in lymph nodes: a study in horses TREVOR J. HEATH* AND STEPHEN A. NIKLES
Department of Anatomy, University of Queensland, Queensland 4072, Australia
(Accepted 10 April 1991) INTRODUCTION
Secondary lymph nodules traditionally appear in the outer cortex, separated from the subcapsular sinus by a variable amount of cortical tissue (Banks, 1986; Raviola, 1986; Brown, Dellmann & Nicander, 1987). Studies in small laboratory animals have revealed that antigenic materials in the lymph entering the subcapsular sinus may rapidly penetrate this cortical tissue and reach the nodule (Nossal & Ada, 1971; Szakal, Holmes & Tew, 1983; Tew, Mandel, Phipps & Szakal, 1983). However, there has been no suggestion from any animals that other sinuses within the node are involved in transport to or from the nodules. Recent studies have shown that the sinus network in horse lymph nodes is more extensive than in other species (Heath & Perkins, 1989; Nikles & Heath, 1991 b). The aim of this study was to define the morphological relationships between these sinuses and the nodules. The study was undertaken using Microfil and methacrylate casts, and by light and electron microscopy.
MATERIALS AND METHODS
Eleven female and 9 male horses, 6-48 months old, were used. All were killed either with an overdose of pentobarbitone, or by humane shooting at a local knackery. These horses were also used either by Heath & Perkins (1989) or by Nikles & Heath (1991 a, b). Up to 10 nodes were taken from each horse, and these were mainly from the following lymph centres: cranial mesenteric, caudal mesenteric, and superficial
inguinal. Methods described by Spalding & Heath (1987) and Nikles & Heath (1991 a) were used to cannulate afferent lymphatic vessels, and to inject either Microfil Rubber Injection Compound (Canton Biomedical Products, Boulder, Colorado), or Batsons 17 Anatomical Corrosion Compound (Paul Valley Industrial Park, Warrington, Pennsylvania) into the lymph sinuses of the node. Methods described by Heath & Perkins (1989) and Nikles & Heath (1991 b) were used to prepare tissues for light microscopy and for scanning electron microscopy. A Philips 505 scanning electron microscope was used. *
Correspondence to Professor T. J. Heath.
40
T. J. HEATH AND S. A. NIKLES RESULTS
The number of secondary nodules within each node varied widely; in some nodes they were sparse, whereas in others nodules were distributed throughout the cortex (Figs 1, 2). There did not appear to be any constant relationship between the number of nodules and the location of the node. The nodules appeared as clear rounded spaces in nodes which had been filled with Microfil and cleared in methyl salicylate (Fig. 3). Their presence was confirmed by cutting the nodes into slices and examining these under a stereomicroscope, and then by studying histological sections prepared from the surface of the slices. The nodules were examined further using a methacrylate-based cast of the lymph sinuses. When the tissue had been digested and the cast broken open to reveal sinuses within the node, scanning electron microscopy revealed rounded spaces similar to those seen in Microfil casts (Figs 3, 4). Examination of the inner surface of these holes revealed a smooth, curving network of filled sinuses, with variable spaces between them (Figs 4, 5). Other nodes were fixed by perfusion of fixative through an afferent lymphatic, and the structure of this network examined in histological sections and by scanning electron microscopy. Virtually all sections through nodules revealed segments of sinuses in the immediate vicinity (Figs 1, 2, 6). In some cases, the sinus-lining cells abutted the capsule of the nodule, but more commonly they were separated by several layers of cells. The nodules at the periphery were adjacent to the subcapsular sinus (Fig. 2), which covered all the cortex. The other types of sinus which were associated with the nodules included those surrounding trabecule, both superficially and deep within the cortex; sinuses between cortical cords of lymphoid parenchyma and the tubule-like extensions of these sinuses into the cortical parenchyma (Figs 1, 2, 6). Some cortical tubular sinuses, which unlike other sinuses had few luminar processes, were also seen in the vicinity of the nodules (Figs 1, 2). DISCUSSION
Secondary lymphoid nodules have been variously referred to as 'follicles' (Tew et al. 1984; van Rooijen, 1987), 'folliculo-nodules' (Sainte-Marie, Peng & Belisle, 1982; Sainte-Marie & Peng, 1986) and 'germinal centres' (Raviola, 1986). In the horse they are surrounded by a network of lymph sinuses. These sinuses include the subcapsular sinus and its extensions around trabeculae, the tubular and tubule-like sinuses deeper in the cortex, and the sinuses between cord-like projections of cortical tissue (Nikles & Heath, 1991 b). These sinuses become filled when casting material is injected through an afferent lymphatic, indicating that they form an interconnecting network throughout the cortex. This contrasts with the situation in the small laboratory animals which have been used to study the movement of antigenic materials within the lymph node. In these animals, the subcapsular sinus appears to be the only sinus that is regularly found in the immediate vicinity of the nodules. However, Sainte-Marie et al. (1982) illustrated infoldings of this sinus near the nodules as one of the variations that may occur in rat lymph nodes. Where the subcapsular sinus forms a continuous sheet, however, a sinus is adjacent to only a small proportion of the nodule surface (SainteMarie et al. 1982; Sainte-Marie & Peng, 1986; van Rooijen, 1987), and separated from it by a subsinus layer of reticular-type cells (Sainte-Marie & Peng, 1985). It appears
~ .,
Nodules and sinuses in lymph nodes
..40
~ ~ ~
~
~
4
~
~
~
~
..
.
A~~~~A
Figs 1, 2. Photomicrographs of sections, stained with haematoxylin and eosin, of the cortex of the horse lymph node. Secondary nodules are located immediately under the subcapsular sinus, and deeper within the cortex. Sinuses in the vicinity of these nodules include the subcapsular sinus (S), trabecular sinuses (T), sinuses between cortical cords (C), and their tubule-like extensions (asterisks) and tubular sinuses (arrows). Fig. 1 x 90; Fig. 2 x 50.
41
42
T. J. HEATH AND S. A. NIKLES
Fig. 3. Photomicrograph of the cut surface of a lymph node which had been filled with Microfili from lymphatic, showing rounded depressions which correspond to nodules. x 20. 5. Figs 4, Scanning electron micrographs of the broken surface of a cast made by injecting Batsons 17 through an afferent lymphatic, showing a network of sinuses surrounding a nodule. The nodule is centrally placed in Figure 4, and occupies the lower half of Figure 5. Fig. 4 x 100; Fig. 5 x 250. Fig. 6. Scanning electron micrograph of the surface of a nodule (N), and an adjacent sinus, which is associated with a trabecula (T). x 200. an afferent
that immune complexes are trapped in the subcapsular sinus and are transported by a group of nonphagocytic cells to the region of the nodules (Szakal et al. 1983; Tew et al. 1984). In horses the nodules, some of which are located relatively deep in the node, are closely associated over much of their surface with an interconnecting network of lymph sinuses. Thus antigenic material carried in the lymph from the tissues would be well placed to influence the activity of nodules irrespective of where they occur in the node. The sinuses in the horse could also provide an important pathway for cell migration within the node, and in this way play a similar role to the 'cortical pathways of migration of circulating lymphocytes' along a reticular fibre network within lymph
Nodules and sinuses in lymph nodes
43
nodes in rats, which was described by Sainte-Marie & Peng (1986). However, no studies appear to have been undertaken on the movement of antigenic materials or cells in the lymph nodes of the horse. In view of the differences in node structure between small laboratory animals and the horse (Heath & Perkins, 1989; Perkins & Heath, 1990; Nikles & Heath, 1991 a, b) it would not be surprising if appreciable differences did exist between these animals in the pathways taken by immunologically important elements within the node. SUMMARY
Secondary lymphoid nodules in lymph nodes of the horse are surrounded by a network of lymph sinuses, including the subcapsular sinus and its extensions around tabeculae, tubular and tubule-like sinuses deeper in the cortex, and sinuses between cord-like projections of cortical tissue. The precise role of this close association between sinuses and nodules in the transport of immune complexes, cells and cytokines is not known. We are grateful to Gary Godbold for help with obtaining specimens, the University of Queensland Electron Microscope Centre for help with electron microscopy, and the Queensland Equine Research Foundation for financial support. REFERENCES
BANKS, W. J. (1986). Applied Veterinary Histology, 2nd ed. Baltimore: Williams & Wilkins. BROWN, E. M., DELLMANN, H.-D. & NICANDER, L. (1987). Lymphatic organs. In Textbook of Veterinary Histology, 3rd ed. (ed. H.-D. Dellmann & E. M. Brown), pp. 164-184. Philadelphia: Lea & Febiger. HEATH, T. J. & PERKINS, N. R. (1989). Pathways between lymph vessels and sinuses in lymph nodes: a study in horses. Anatomical Record 223, 420424. NIKLES, S. A. & HEATH, T. J. (1991 a). Pathways of lymph flow from the intestine of the horse. Anatomical Record 229, 521-524. NIKLES, S. A. & HEATH, T. J. (1991 b). Pathways of lymph flow through intestinal lymph nodes in the horse. Anatomical Record, in press. NOSSAL, G. J. V. & ADA, G. L. (1971). Microscopic and electronmicroscopic distribution of antigen in lymphoid organs. In Antigens, Lymphoid Cells and the Immune Response, pp. 107-141. New York: Academic Press. PERKINS, N. R. & HEATH, T. J. (1990). Pathways of lymph flow from the superficial tissues in the legs of horses. Research in Veterinary Science 48, 119-123. RAVIOLA, E. (1986). The immune system. In Bloom and Fawcett, A Textbook of Histology (ed. D. W. Fawcett), pp. 406-435. Philadelphia: Saunders. SAINTE-MAREE, G. & PENG, F.-S. (1985). Evidence for the existence of a subsinus layer of the peripheral cortex in the lymph node of the rat. Cell and Tissue Research 239, 37-42. SAINTE-MARIE, G. & PENG, F.-S. (1986). Diffusion of lymph-carried antigen in the fiber network of the lymph node of the rat. Cell and Tissue Research 245, 481-486. SAINTE-MAREE, G., PENG, F.-S. & BESLISLE, C. (1982). Overall architecture and pattern of lymph flow in the rat lymph node. American Journal of Anatomy 184, 275-309. SPALDING, H. J. & HEATH, T. J. (1987). Pathways of lymph flow through superficial inguinal lymph nodes in the pig. Anatomical Record 217, 188-195. SZAKAL, A. K., HOLMES, K. L. & TEw, J. G. (1983). Transport of immune complexes from the subcapsular sinus to lymph node follicles on the surface of nonphagocytic cells, including cells with dendritic morphology. Journal of Immunology 131, 1714-1727. TEW, J. G., MANDEL, T. E., PHIpps, R. P. & SZAKAL, A. K. (1984). Tissue localization and retention of antigen in relation to the immune response. American Journal of Anatomy 170, 407-420. VAN ROOIJEN, N. (1987). The 'in situ' immune response in lymph nodes: a review. Anatomical Record 218, 359-364.
